Synthesis of sphingolipids with an ω-Esterified long acyl chain, ceramide components of the human epidermis

Article abstract of DOI:10.1271/bbb.120333

Esterified ceramides, (2S,3R,4E)-2-[(300-stearoyloxy) triacontanamido] octadec-4-ene-1,3-diol (2) and (2S,3R,4E,6R)-2-[(300-stearoyloxy) triacontanamido]octadec- 4-ene-1,3,6-triol (4), are minor components of the human stratum corneum. We synthesized thes

Full text of DOI:10.1271/bbb.120333

Biosci. Biotechnol. Biochem., 76 (9), 1715–1720, 2012
Synthesis of Sphingolipids with an !-Esterified Long Acyl Chain,
*
Ceramide Components of the Human Epidermis
y
Takuya TASHIRO and Kenji MORI
Glycosphingolipid Synthesis Group, Laboratory for Immune Regulation, Research Center for Allergy and Immunology,
RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Received April 27, 2012; Accepted May 30, 2012; Online Publication, September 7, 2012
[doi:10.1271/bbb.120333]
Esterified
ceramides,
(2S,3R,4E)-2-[(300-stearo-
OH
yloxy)triacontanamido]octadec-4-ene-1,3-diol (2) and
(2S,3R,4E,6R)-2-[(300-stearoyloxy)triacontanamido]octa-
dec-4-ene-1,3,6-triol (4), are minor components of the
human stratum corneum. We synthesized these ceram-
ides by employing olefin cross metathesis as the key
reaction for constructing their !-hydroxytriacontanoyl
part (13).
HO
(CH2)10Me
NH
(CH2)29
ceramide1
O
R
O
O
)
(CH2)4Me
1: R = -(CH2 7
2: R = -(CH2)16Me
OH
Key words: ceramide; olefin cross metathesis; epider-
mis; sphingolipid; stratum corneum
HO
(CH2)10Me
NH
OH
O
R
The stratum corneum is the upper layer of the
epidermis. It protects the body from loss of water and
against intrusion of environmental damaging factors.2,3)
Ceramides are the major lipid components, accounting
for 30–40% by weight of the mammalian stratum
corneum.2) To date, nine subclasses of free extractable
ceramides have been identified from the human stratum
corneum.4–8) Among these, ceramides 1, 4, and 9 are
structurally unique sphingolipids, and respectively com-
prise 8%, 5%, and 6% of free and extractable ceram-
ides.4) They have a long acyl chain possessing an !-
hydroxy group esterified with a fatty acid like linoleic
acid. It has been proposed that these esterified ceramides
function as molecular rivets in locking together the
multiple intercellular lamellae with their alkyl chains in
the stratum corneum.9,10)
Esterified ceramides 1, 3, and 5 (Fig. 1) are the major
respective components of ceramides 1, 4, and 9.4–8) They
have an !-linoleoyloxy acyl group, but it has not yet been
clarified why the linoleoyl group is so abundantly
distributed in the epidermal ceramides. We have reported
the synthesis of 1 and 3, and confirmed their structures as
shown.11,12) Due to the fragility of the 1,4-diene structure
of the linoleoyl part, ceramides 1 and 3 are unstable.
Therefore Dr. S. Hamanaka, a dermatologist, asked us to
synthesize !-stearoylated ceramides 2 and 4 as stable
standard samples for a dermatological study. It is known
that 2 and 4 are natural minor components of ceramides 1
and 4.4) We reported in 1992 a synthesis of !-stearoyloxy
ceramide with an icosasphingosine (C20) chain.13) How-
ever, the synthesis of its sphingosine (C18)-type ceram-
ide (2) and 6-hydroxysphingosine-type one (4) have not
previously been reported.
O
(CH2)29
O
ceramide4
)
(CH2)4Me
3: R = -(CH2 7
4: R = -(CH2)16Me
OH
HO
(CH2)10Me
(CH2)7
NH OH
O
(CH2)4Me
O
(CH2)29
O
ceramide 9 (5)
Fig. 1. Structures of Esterified Ceramides 1–5, Components of the
Human Stratum Corneum.
overall yields of 2 and 4 were 17% and 12%, based on 6.
We employed olefin cross metathesis with Grubbs’s
first-generation catalyst as the key reaction to construct
their !-hydroxytriacontanoic acid part.14–16)
Results and Discussion
Our previous synthesis of 1 and 3,11,12) including
icosasphingosine-type ceramide 1,13) employed the
Wittig reaction for constructing their !-hydroxytriacon-
tanoyl part.17) Although the overall yield is generally
very high, the Wittig reaction requires the cumbersome
preparation of both an ylide and an aldehyde, and makes
the synthetic pathway redundant. We chose in this
present synthesis the olefin cross metathesis reaction to
assemble the long acyl chain part.14–16)
Figure 2 summarizes the retrosynthetic analysis of 2
and 4. We considered that the synthesis of the target
ceramides, including 1 and 3, could be achieved by a
two-step conversion, acylation and deprotection, from
We synthesized 2 and 4 from 15-pentadecanolide (6)
in 9 steps as shown in Schemes 1 and 2. The respective
*
y
Synthesis of sphingosine relatives, Part 35. See Ref. 1 for Part 34.
To whom correspondence should be addressed. Fax: +81-48-467-9381; E-mail: kjk-mori@arion.ocn.ne.jp

1716
T. TASHIRO and K. MORI
OPG
PGO
(CH2)10Me
Cross
OPG
NH2
R
2 or 4
PGO
(CH2)10Me
B: R = H or OPG
NH
R
HO(CH2)14
O
(CH2)29OH
O
metathesis
D
HO
(CH2)29OAc
A: R = H or OPG
C
(CH2)14OAc
PG = protective group
E
Fig. 2. Synthetic Plan.
O
O
a
c
e
HO(CH2)14
HO(CH2)30OAc
(CH2)14OR
7 + 8
(CH2)14OAc
9 (E/Z = ca. 4:1)
7 (= D): R = H
8 (= E): R = Ac
12
b
d
+
6
AcO(CH2)14
PCy3
Ru
(CH2)14OAc
Cl
Cl
10 (E/Z = ca. 3:1)
Ph
PCy3
+
f
HO(CH2)14
Grubbs I catalyst
Cy = cyclohexyl
HO2C(CH2)29OAc
(CH2)14OH
11 (E/Z = ca. 4:1)
13 (= C)
Scheme 1. Synthesis of !-Acetoxytriacontanoic Acid (13).
Reagents and conditions: (a) (i-Bu)2AlH, toluene, ꢁ78 ꢀC, 30 min; then Ph3P=CH2 [prepared from Ph3PMeBr and LiN(SiMe3)2], THF,
ꢁ78 ꢀC to room temperature (64%); (b) Ac2O, pyridine, room temperature (99%); (c) 5 mol % Grubbs I catalyst [(Cy3P)2Ru(=CHPh)Cl2],
CH2Cl2, reflux, 3 h (47% for 9, 22% for 10, 23% for 11); (d) 5 mol % Grubbs I catalyst, CH2Cl2, reflux, 3 h (45% for 9, 23% for 10, 20% for 11);
(e) H2, 10% Pd–C, THF, room temperature, 15 h (93%); (f) Jones CrO3 reagent, acetone, 60 ꢀC (96%).
intermediate A. Alcohol A could be prepared by
condensing amine B with acid C. To construct C, we
planned to employ the olefin cross metathesis reaction
with Grubbs’s first-generation catalyst between D and E
which could be readily prepared from commercially
available 15-pentadecanolide (6).
9 yielded 12 (93%) which was then oxidized with the
Jones chromic acid reagent to give acid 13 in a 96%
yield.
Many methods have been published to construct
the sphingosine chain.18,19) We prepared it in the
present synthesis from commercially available and
inexpensive phytosphingosine 1420) via Kim’s azide 15
(Scheme 2).21) Protection of the 3-hydroxy group of 15
as a tert-butyldimethylsilyl (TBS) ether provided 16.
Reduction of 16 with trimethylphosphine and subse-
quent alkaline treatment gave amine 17a (= B) in an
88% yield. Condensation of 17a with acid 13 afforded
18a (98%), and deacetylation of 18a yielded the key
intermediate, alcohol 19a (= A), in a 90% yield.
Acylation of the !-hydroxyl group of 19a with stearoyl
chloride gave 20a (91%) which was then desilylated
with tetrabutylammonium fluoride (TBAF) to furnish
one of the desired !-stearoyloxy ceramides, 2. The
overall yield was 17% in 9 steps from 6.
Synthesis of a 6-hydroxysphingosine chain like 17b
has been reported by three groups.12,22–24) Another target
ceramide (4) was synthesized from amine 17b, which
had been prepared according to the method in our
previous report,12) in the same manner as that just
described. The overall yield of 4 was 12% based on 6 in
9 steps.
Scheme 1 shows how !-acetoxytriacontanoic acid 13
(= C) was synthesized from 6. Lactone 6 was converted
to alken-1-ol 7 (= D) via the corresponding hemiacetal
by reduction with diisobutylaluminum hydride and
subsequent treatment with the Wittig reagent in a one-
pot procedure (64%). Acetylation of 7 gave 8 (= E) in a
99% yield. The olefin cross metathesis reaction between
7 and 8 (a 1:1 mixture) in the presence of 5 mol % of
Grubbs’s first-generation catalyst gave desired olefin 9
(47%, E/Z = ca. 4:1, determined by a 500 MHz 1H-
NMR analysis) together with diacetate 10 (22%) and
diol 11 (23%).14–16) These compounds were separated by
column chromatography, and the olefin cross metathesis
reaction of a 1:1 mixture of separated 10 and 11
provided three kinds of olefin, 9 (45%), 10 (23%), and
11 (20%). The reaction mixture needed to be loaded into
a chromatography column while it was hot after stirring
under reflux for 3 h to achieve equilibrium of the
metathesis reaction. Purification after cooling to room
temperature resulted in respective isolated yields for 9,
10 and 11 of 39% (E/Z = ca. 4:1), 23% (E/Z = ca. 4:1)
and 29% (E/Z = ca. 9:1). The solubility of 11 in
dichloromethane was lower than that of 9 and 10, and it
separated out from the solution when it was cooled to
room temperature. Therefore, 11 was regenerated from 9
through the metathesis reaction in the solution. This
decreased the yield of 9, and 11 was obtained in more
than the theoretical yield in this case. Hydrogenation of
Synthesized 2 and 4 were hardly soluble in CHCl3,
although it has been reported that 1 and 3 were soluble
in CHCl3 even at room temperature.11,12) The physical
properties of these esterified ceramides might be
influenced by the nature of their !-acyloxy groups.
The respective melting points of the synthesized !-
stearoyloxy ceramides (2 and 4) were 94–95 ꢀC and
109–111 ꢀC. The bioactivity of these ceramides is now

Synthesis of Ceramides of the Human Epidermis
1717
OTBS
OH
OR
Ref.21
OTBS
b
TBDPSO
(CH2)12Me
+
HO2C(CH2)29OAc
HO
(CH2)12Me
TBDPSO
a
(CH2)12Me
NH2
17a (= B)
N3 OH
N3
13
15: R = H
14
16: R = TBS
OH
c
f
HO
(CH2)12Me
O
TBDPSO
(CH2)12Me
NH
NH
(CH2)29OR
18a: R = Ac
19a: R = H (=A)
20a: R = CO(CH2)16Me
O
(CH2)29
O
(CH2)16Me
O
2
d
e
OH
OTBS
Similarly
TBSO
(CH2)11Me
HO O
(CH2)29 (CH2)16Me
HO
(CH2)11Me
OTBS
17b12 (= B)
+
13
+ stearoyl chloride
NH
NH2
O
O
4
Scheme 2. Synthesis of Esterified Ceramides 2 and 4.
Reagents and conditions: (a) TBSOTf, 2,6-lutidine, CH2Cl2, 0 ꢀC (quant.); (b) PMe3, THF, room temperature, 17 h; then 1.0 M NaOH aq.,
room temperature, 2 h (88%); (c) 13, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, (i-Pr)2NEt, room
temperature, 19 h (98%); (d) K2CO3, MeOH, CH2Cl2, room temperature, 18 h (90%); (e) stearoyl chloride, pyridine, CHCl3, room temperature,
45 min (91%); (f) TBAF, THF, room temperature, 24 h (80%).
the mixture was stirred for 30 min. The mixture was extracted with
EtOAc, and the combined organic phase was successively washed with
under investigation, and will be reported in due course
by Dr. S. Hamanaka.
water, a saturated aqueous NaHCO3 solution and brine, dried with
MgSO4, and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (50 g, hexane/EtOAc = 10:1) to
Conclusion
give 7 (928 mg, 64%) as colorless needles. IR and 1H-NMR spectra
were in accordance with those reported in ref. 25. Mp 33–35 ꢀC
(ref. 25, oil); IR ꢁmax (KBr) cmꢁ1: 3280 (br m, OH), 3080 (w), 1645
(m, C=C), 990 (m), 915 (s), 760 (s); 1H-NMR ꢀH (500 MHz, CDCl3):
1.20 (1H, br s), 1.23–1.34 (20H, m), 1.36 (2H, quint., J ¼ 7:0 Hz), 1.57
(2H, quint., J ¼ 7:0 Hz), 2.04 (2H, dddt, J ¼ 1:0, 1.5, 7.0, 7.0 Hz),
3.64 (2H, t, J ¼ 6:5 Hz), 4.93 (1H, ddt, J ¼ 2:0, 10, 1.0 Hz), 4.99 (1H,
ddt, J ¼ 2:0, 17, 1.5 Hz), 5.82 (1H, ddt, J ¼ 10, 17, 7.0 Hz). HRMS
(EI+): calcd. for C16H30 [ðM ꢁ H2OÞþ], 222.2348; found, 222.2345.
The minor ceramide components (2 and 4) of the
human stratum corneum were synthesized from sphin-
gosine bases (17a and 17b) and !-acetoxytriacontanoic
acid (13). The latter was prepared by olefin cross
metathesis between hexadec-15-en-1-ol (7) and its
acetate (8), employing Grubbs’s first-generation cata-
lyst. The preparation of 7 was accomplished by a one-
pot reduction/Wittig reaction procedure from 15-penta-
decanolide (6).
Hexadec-15-enyl acetate (8). To a stirred solution of 7 (819 mg,
3.41 mmol) in pyridine (15 mL), Ac2O (644 mL, 6.81 mmol) was added
at 0 ꢀC. After stirring at room temperature for 15 h, the reaction was
quenched with water. The mixture was extracted with EtOAc, and the
separated organic phase was successively washed with 1 M hydro-
chloric acid, water, a saturated aqueous NaHCO3 solution and brine,
dried with MgSO4, and concentrated in vacuo. The residue was
chromatographed on silica gel (20 g, hexane/EtOAc = 20:1) to give 8
(952 mg, 99%) as a colorless oil, nD 1.4477. IR ꢁmax (film) cmꢁ1: 3080
(w), 1740 (s, C=O), 1640 (m, C=C), 1240 (br s, C–O), 1040 (br m,
C–O), 990 (w), 910 (m); 1H-NMR ꢀH (500 MHz, CDCl3): 1.22–1.35
(20H, m), 1.36 (2H, quint., J ¼ 7:0 Hz), 1.62 (2H, quint., J ¼ 7:0 Hz),
2.03 (2H, br q, J ¼ 7:0 Hz), 2.05 (3H, s), 4.05 (2H, t, J ¼ 7:0 Hz), 4.93
(1H, ddt, J ¼ 2:0, 10, 1.0 Hz), 4.99 (1H, ddt, J ¼ 2:0, 17, 1.5 Hz), 5.82
(1H, ddt, J ¼ 10, 17, 7.0 Hz). HRMS (EI+): calcd. for C18H34O [Mþ],
282.2559; found, 282.2551.
Experimental
Refractive indices (nD) were measured with an Atago 1T
refractometer. Melting point (mp) data were recorded with Yanaco
MP-S3 melting point measuring apparatus and are uncorrected. Optical
rotation values were measured with a Jasco P-1010 polarimeter, and IR
spectra were measured with a Jasco FT/IR-460 plus spectrometer. 1H-
NMR spectra (TMS at ꢀH ¼ 0:00 or pyridine at ꢀH ¼ 7:55 as the
internal standards) and 13C-NMR spectra (pyridine at 135.5 as the
internal standard) were recorded with a Varian VNMRS-500 spec-
trometer. High-resolution mass spectrometry (HRMS) was performed
with a Jeol JMS-700 [electron ionization (EI)] or Waters MALDI
SYNAPTꢀ G2 HDMS [electrospray ionization (ESI)] mass spec-
trometer. Column chromatography was performed with Merck
Kieselgel 60 Art 1.07734.
(15EZ)-30-Hydroxytriacont-15-enyl acetate (9).
Method A: A solution of 7 (524 mg, 2.18 mmol), 8 (615 mg,
2.18 mmol), and (Cy3P)2Ru(=CHPh)Cl2 (Aldrich, Grubbs’s first-
generation catalyst, 106 mg, 129 mmol, 5.6 mol % to 7) in dry and
degassed CH2Cl2 (20 mL) was stirred under reflux for 3 h in an argon
atmosphere. The reaction mixture was then diluted with hot CHCl3 and
loaded into a chromatographic column of silica gel (80 g) while the
mixture was hot. Purification by column chromatography was then
performed at room temperature.
Hexadec-15-en-1-ol (7). To a stirred suspension of methyltriphe-
nylphosphonium bromide (4.47 g, 12.5 mmol) in dry THF (20 mL) was
added a solution of lithium bis(trimethylsilyl)amide (LHMDS, 1.0 M in
THF, 12.5 mL, 12.5 mmol) at 0 ꢀC. The mixture was stirred for 2 h at
0 ꢀC to prepare a solution of methylenetriphenylphosphorane.
To a stirred solution of 6 (1.44 g, 5.99 mmol) in dry toluene (30 mL)
at ꢁ78 ꢀC, a solution of diisobutylaluminum hydride (DIBAL-H,
1.02 M in toluene, 6.5 mL, 6.6 mmol) was slowly added over 30 min.
(15EZ)-Diacetoxytriacont-15-ene (10, 263 mg, 22%) was eluted
with CHCl3/MeOH=150:1 as a pale brown solid (E:Z = ca. 3:1). Mp
56–60 ꢀC; IR ꢁmax (KBr) cmꢁ1: 1740 (br s, C=O), 1250 (br s), 1060
(m, C–O), 1040 (m, C–O), 965 (s); 1H-NMR ꢀH (500 MHz, CDCl3):
1.20–1.37 (44H, m), 1.61 (4H, quint., J ¼ 7:0 Hz), 1.96 (3H, m), 2.01
After stirring at ꢁ78 ꢀC for 30 min,
a yellow solution of the
phosphorous ylide just prepared was slowly added over 10 min. The
mixture was gradually warmed to room temperature while stirring for
17 h. The reaction was then quenched with 1 M hydrochloric acid, and

1718
T. TASHIRO and K. MORI
(1H, m), 2.05 (6H, s), 4.05 (4H, t, J ¼ 7:0 Hz), 5.35 (0.5H, tt, J ¼ 1:0,
4.5 Hz), 5.38 (1.5H, tt, J ¼ 1:5, 3.5 Hz). HRMS (EI+): calcd. for
C34H64O4 [Mþ], 536.4805; found, 536.4805.
700 (s); 1H-NMR ꢀH (500 MHz, CDCl3): 0.01 (3H, s), 0.03 (3H, s),
0.83 (9H, s), 0.89 (3H, t, J ¼ 7:0 Hz), 1.08 (9H, s), 1.23–1.36 (22H,
m), 2.00 (2H, dr q, J ¼ 7:0 Hz), 3.50 (1H, dt, J ¼ 5:5, 7.0 Hz), 3.64
(1H, dd, J ¼ 7:0, 11 Hz), 3.71 (1H, dd, J ¼ 5:5, 11 Hz), 4.20 (1H, dd,
J ¼ 5:5, 7.0 Hz), 5.38 (1H, ddt, J ¼ 1:5, 7.0, 15 Hz), 5.60 (1H, dt,
J ¼ 7:0, 15 Hz), 7.37–7.46 (6H, m), 7.67 (4H, t, J ¼ 7:0 Hz). HRMS
(ESI+): calcd. for C40H67N3O2Si2Na [ðM þ NaÞþ], 700.4670; found,
700.4658.
(15EZ)-30-Hydroxytriacont-15-enyl acetate (9, 510 mg, 47%) was
eluted with CHCl3/MeOH=150:2 as pale brown powder (E:Z = ca.
4:1). Mp 66–69 ꢀC; IR ꢁmax (KBr) cmꢁ1: 3280 (br m, OH), 1740 (br s,
C=O), 1255 (br s), 1060 (m, C–O), 1045 (m, C–O), 960 (m); 1H-NMR
ꢀH (500 MHz, CDCl3): 1.21–1.37 (45H, m), 1.56 (2H, quint.,
J ¼ 7:0 Hz), 1.62 (2H, quint., J ¼ 7:0 Hz), 1.96 (3.2H, m), 2.01
(0.8H, m), 2.05 (3H, s), 3.64 (2H, t, J ¼ 7:0 Hz), 4.05 (2H, t,
J ¼ 7:0 Hz), 5.35 (0.4H, tt, J ¼ 1:0, 4.5 Hz), 5.38 (1.6H, tt, J ¼ 1:5,
4.0 Hz). HRMS (EI+): calcd. for C32H62O3 [Mþ], 494.4699; found,
494.4697.
(2S,3R,4E)-2-Amino-3-O-(tert-butyldimethylsilyl)-1-O-(tert-butyldi-
phenylsilyl)octadec-4-ene-1,3-diol (17a). To a stirred solution of 16
(447 mg, 0.659 mmol) in dry THF (15 mL), a solution of trimethyl-
phosphine (1.0 M in THF, 3.3 mL, 3.3 mmol) was added at room
temperature. After stirring at room temperature for 17 h, an aqueous
solution of NaOH (1.0 M, 6.6 mL, 6.6 mmol) was added to the mixture.
The resulting mixture was stirred at room temperature for 2 h. The
mixture was then poured into water and extracted with EtOAc. The
separated organic phase was successively washed with water, a
saturated aqueous NaHCO3 solution and brine, dried with K2CO3, and
concentrated in vacuo. The residue was purified by column chroma-
tography on silica gel (30 g, hexane/EtOAc = 9:1) to give 17a
(15EZ)-Triacont-15-ene-1,30-diol (11, 223 mg, 23%) was eluted
with CHCl3/MeOH=150:3 as pale brown powder (E:Z = ca. 4:1). Mp
95–98 ꢀC; IR ꢁmax (KBr) cmꢁ1: 3280 (br m, OH), 1060 (m, C–O), 960
(m); 1H-NMR ꢀH (500 MHz, CDCl3, 40 ꢀC): 1.19 (2H, br s), 1.20–1.38
(44H, m), 1.56 (4H, quint., J ¼ 6:5 Hz), 1.96 (3.2H, m), 2.01 (0.8H,
m), 3.63 (4H, t, J ¼ 6:5 Hz), 5.35 (0.4H, br t, J ¼ 4:5 Hz), 5.38 (1.6H,
tt, J ¼ 1:5, 4.0 Hz). HRMS (EI+): calcd. for C30H60O2 [Mþ],
452.4593; found, 452.4589.
23
23
Method B: A suspension of 10 (277 mg, 0.516 mmol), 11 (234 mg,
0.517 mmol), and Grubbs’s first-generation catalyst (Aldrich, 22 mg,
0.027 mmol, 5.2 mol % to 10) in dry and degassed CH2Cl2 (20 mL) was
stirred under reflux for 3 h in an argon atmosphere. The mixture was
then diluted with hot CHCl3 and purified by column chromatography
on silica gel (30 g, CHCl3/MeOH = 100:1, 100:2, and 100:3 for 10, 9,
and 11, respectively) with the same procedure as that just described to
give 10 (129 mg, 23%, E/Z = ca. 4:1), 9 (288 mg, 45%, E/Z=ca. 3:1)
and 11 (95 mg, 20%, E/Z = ca. 5:1).
(378 mg, 88%) as a colorless oil, nD 1.5062; ½ꢂꢂD ꢁ3:36 (c 1.00,
CHCl3). IR ꢁmax (film) cmꢁ1: 3390 (w, NH), 1660 (w, C=C), 1590
(w), 1255 (s, t-Bu, Si-Me), 1115 (s, C–O), 1080 (br s, C–O), 970 (m),
835 (br s), 775 (m), 740 (m), 700 (s); 1H-NMR ꢀH (500 MHz, CDCl3):
ꢁ0:04 (3H, s), ꢁ0:03 (3H, s), 0.80 (9H, s), 0.88 (3H, t, J ¼ 7:0 Hz),
1.06 (9H, s), 1.22–1.36 (22H, m), 1.50 (2H, br s), 2.00 (2H, dr q,
J ¼ 7:0 Hz), 2.89 (1H, dt, J ¼ 7:0, 5.0 Hz), 3.57 (1H, dd, J ¼ 7:0,
10 Hz), 3.73 (1H, dd, J ¼ 5:0, 10 Hz), 4.07 (1H, t, J ¼ 7:0 Hz), 5.34
(1H, br dd, J ¼ 7:0, 15 Hz), 5.60 (1H, dt, J ¼ 15, 7.0 Hz), 7.34–7.45
(6H, m), 7.63–7.67 (4H, m). HRMS (ESI+): calcd. for C40H70NO2Si2
[ðM þ HÞþ], 652.4945; found, 652.4926.
30-Hydroxytriacontyl acetate (12). To a stirred solution of 9
(498 mg, 1.01 mmol) in THF (40 mL), 10% Pd–C (Kawaken, 84 mg)
was added. After stirring at room temperature for 15 h in a hydrogen
atmosphere, the mixture was filtered and then concentrated in vacuo.
The residue was purified by column chromatography on silica gel
(20 g, hexane/EtOAc = 10:1) to give 12 (467 mg, 93%) as colorless
powder. Mp 84–86 ꢀC; IR ꢁmax (KBr) cmꢁ1: 3280 (br m, OH), 1735 (s,
C=O), 1260 (s), 1055 (m, C–O); 1H-NMR ꢀH (500 MHz, CDCl3):
1.20–1.38 (53H, m), 1.57 (2H, quint., J ¼ 7:0 Hz), 1.62 (2H, quint.,
J ¼ 7:0 Hz), 2.05 (3H, s), 3.64 (2H, t, J ¼ 7:0 Hz), 4.05 (2H, t,
J ¼ 7:0 Hz). HRMS (EI+): calcd. for C32H64O3 [Mþ], 496.4855;
found, 496.4847.
(2S,3R,4E)-2-[(300-Acetoxy)triacontanamido]-3-O-(tert-butyldime-
thylsilyl)-1-O-(tert-butyldiphenylsilyl)octadec-4-ene-1,3-diol (18a). To
a stirred mixture of 17a (302 mg, 0.463 mmol) and 13 (235 mg,
0.460 mmol) in dry CH2Cl2 (10 mL), 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDC, 180 mg, 0.939 mmol), 1-
hydroxybenzotriazole (HOBt, 64 mg, 0.47 mmol), and (i-Pr)2NEt
(162 mL, 0.930 mmol) were added at room temperature. After stirring
at room temperature for 19 h, the mixture was poured into water and
extracted with EtOAc. The separated organic phase was successively
washed with water, a saturated aqueous NaHCO3 solution and brine,
dried with MgSO4, and concentrated in vacuo. The residue was
purified by column chromatography on silica gel (30 g, hexane/
EtOAc = 9:1) to give 18a (522 mg, 98%) as a colorless solid. Mp 52–
30-Acetoxytriacontanoic acid (13). To a stirred suspension of 12
(422 mg, 0.849 mmol) in dry acetone (30 mL), a solution of Jones CrO3
(2.67 M, 953 mL, 2.54 mmol) was added in one portion at room
temperature. After stirring at 60 ꢀC for 30 min, the mixture was diluted
with water. The precipitate was collected by filtration and then washed
with water. After air-drying, 415 mg (96%) of 13 was obtained as
colorless powder and was used in the next step without further
purification. The analytical sample was obtained by column chroma-
tography on silica gel (CHCl3/MeOH = 50:2). Mp 90–93 ꢀC; IR ꢁmax
(KBr) cmꢁ1: 3000 (br s, OH), 1740 (s, C=O), 1710 (br s, C=O), 1250
(br s), 1045 (br m); 1H-NMR ꢀH (500 MHz, CDCl3): 1.22–1.37 (50H,
m), 1.57 (1H, br s), 1.58–1.67 (4H, m), 2.05 (3H, s), 2.35 (2H, t,
J ¼ 7:0 Hz), 4.05 (2H, t, J ¼ 7:0 Hz). HRMS (ESI+): calcd. for
C32H62O4Na [ðM þ NaÞþ], 533.4546; found, 533.4546.
23
54 ꢀC; ½ꢂꢂD ꢁ3:74 (c 1.00, CHCl3). IR ꢁmax (KBr) cmꢁ1: 1735 (br m,
C=O), 1680 (s, C=O), 1660 (w, C=C), 1525 (m), 1260 (br m, t-Bu,
Si-Me), 1120 (s, C–O), 1080 (br s, C–O), 1045 (br s, C–O), 970 (m),
860 (m), 840 (m), 760 (s), 700 (m); 1H-NMR ꢀH (500 MHz, CDCl3):
ꢁ0:02 (3H, s), ꢁ0:01 (3H, s), 0.82 (9H, s), 0.88 (3H, t, J ¼ 7:0 Hz),
1.07 (9H, s), 1.21–1.36 (74H, m), 1.62 (2H, quint., J ¼ 7:0 Hz), 1.95
(2H, q, J ¼ 7:0 Hz), 2.00 (2H, dt, J ¼ 5:0, 7.0 Hz), 2.05 (3H, s), 3.70
(1H, dd, J ¼ 5:0, 11 Hz), 3.88 (1H, dd, J ¼ 5:0, 11 Hz), 4.01 (1H, m),
4.05 (2H, t, J ¼ 7:0 Hz), 4.30 (1H, br t, J ¼ 7:0 Hz), 5.34 (1H, br dd,
J ¼ 7:0, 15 Hz), 5.43 (1H, d, J ¼ 8:5 Hz), 5.58 (1H, br dt, J ¼ 15,
7.0 Hz), 7.35–7.46 (6H, m), 7.63 (4H, t, J ¼ 8:0 Hz). HRMS (ESI+):
calcd. for C72H130NO5Si2 [ðM þ HÞþ], 1144.9488; found, 1144.9508.
(2S,3R,4E)-2-Azido-3-O-(tert-butyldimethylsilyl)-1-O-(tert-butyldi-
phenylsilyl)octadec-4-ene-1,3-diol (16). To a stirred solution of 1521)
(574 mg, 1.02 mmol) and 2,6-lutidine (355 mL, 3.05 mmol) in dry
CH2Cl2 (10 mL), tert-butyldimethylsilyl trifluoromethanesulfonate
(TBSOTf, 351 mL, 1.53 mmol) was added at 0 ꢀC. After stirring at
0 ꢀC for 15 min, the reaction was quenched with MeOH (0.5 mL), and
the mixture was concentrated in vacuo. The residue was diluted with
EtOAc, and successively washed with a saturated aqueous NaHCO3
solution, water and brine, dried with MgSO4, and concentrated in
vacuo. The residue was purified by column chromatography on silica
gel (30 g, hexane/EtOAc = 50:1) to give 16 (711 mg, quant.) as a
(2S,3R,4E,6R)-2-[(300-Acetoxy)triacontanamido]-1,3,6-tris-O-(tert-
butyldimethylsilyl)octadec-4-ene-1,3,6-triol (18b). In the same manner
as that just described, 17b12) (152 mg, 0.231 mmol) was converted to
24
18b (265 mg, quant.) as colorless wax. Mp 40–43 ꢀC; ½ꢂꢂD ꢁ1:23
(c 1.02, CHCl3). IR ꢁmax (film) cmꢁ1: 3440 (w, NH), 3360 (br m, NH),
1745 (s, C=O), 1675 (w, C=C), 1660 (br s, C=O), 1510 (br m), 1255
(br s, t-Bu, Si-Me), 1085 (br s, C–O), 970 (w), 840 (br s), 775 (s);
1H-NMR ꢀH (500 MHz, CDCl3): 0.010 (3H, s), 0.012 (3H, s), 0.03
(6H, s), 0.04 (3H, s), 0.05 (3H, s), 0.877 (3H, t, J ¼ 7:0 Hz), 0.881
(9H, s), 0.884 (9H, s), 0.89 (9H, s), 1.20–1.38 (70H, m), 1.40–1.52
(2H, m), 1.52–1.64 (4H, m), 2.05 (3H, s), 2.12 (2H, dt, J ¼ 2:0,
7.5 Hz), 3.54 (1H, dd, J ¼ 6:0, 10 Hz), 3.77 (1H, dd, J ¼ 5:5, 10 Hz),
3.98 (1H, m), 4.05 (2H, t, J ¼ 7:0 Hz), 4.12 (1H, q, J ¼ 6:0 Hz),
4.42 (1H, t, J ¼ 6:0 Hz), 5.47 (1H, d, J ¼ 7:5 Hz), 5.60 (1H, ddd,
23
colorless oil, nD23 1.5040; ½ꢂꢂD ꢁ11:8 (c 1.03, CHCl3). IR ꢁmax (film)
cmꢁ1: 2110 (s, N3), 1670 (w, C=C), 1590 (w), 1255 (br m, t-Bu, Si-
Me), 1015 (br s, C–O), 1070 (br s, C–O), 970 (m), 840 (br s), 780 (m),

Synthesis of Ceramides of the Human Epidermis
1719
23
J ¼ 1:0, 6.0, 16 Hz), 5.70 (1H, ddd, J ¼ 1:0, 6.0, 16 Hz). HRMS
(ESI+): calcd. for C68H139NO6Si3Na [ðM þ NaÞþ], 1172.9808; found,
1172.9775.
½ꢂꢂD ꢁ0:96 (c 1.17, CHCl3). IR ꢁmax (KBr) cmꢁ1: 3440 (w, NH),
3360 (m, NH), 1740 (s, C=O), 1675 (w, C=C), 1655 (br s, C=O),
1540 (m), 1255 (s, t-Bu, Si-Me), 1180 (s, C–O), 1100 (br s, C–O), 975
(w), 840 (br s), 780 (s); 1H-NMR ꢀH (500 MHz, CDCl3): 0.012 (3H, s),
0.014 (3H, s), 0.036 (6H, s), 0.043 (3H, s), 0.05 (3H, s), 0.880 (6H, t,
J ¼ 7:5 Hz), 0.884 (9H, s), 0.887 (9H, s), 0.890 (9H, s), 1.20–1.38
(100H, m), 1.40–1.52 (2H, m), 1.58–1.64 (4H, m), 2.13 (2H, dt,
J ¼ 2:0, 7.5 Hz), 2.29 (2H, t, J ¼ 7:0 Hz), 3.54 (1H, dd, J ¼ 6:0,
11 Hz), 3.78 (1H, dd, J ¼ 5:5, 11 Hz), 3.98 (1H, m), 4.05 (2H, t,
J ¼ 7:0 Hz), 4.12 (1H, q, J ¼ 5:5 Hz), 4.42 (1H, t, J ¼ 5:5 Hz), 5.47
(1H, d, J ¼ 8:0 Hz), 5.60 (1H, ddd, J ¼ 1:0, 5.5, 16 Hz), 5.71 (1H, ddd,
J ¼ 1:0, 5.5, 16 Hz). HRMS (ESI+): calcd. for C84H171NO6Si3Na
[ðM þ NaÞþ], 1397.2312; found, 1397.2297.
(2S,3R,4E)-3-O-(tert-Butyldimethylsilyl)-1-O-(tert-butyldiphenylsilyl)-
2-[(300-hydroxy)triacontanamido]octadec-4-ene-1,3-diol (19a). To a
stirred solution of 18a (202 mg, 0.176 mmol) in MeOH–CH2Cl2 (1:1,
20 mL), K2CO3 (50 mg, 0.36 mmol) was added at room temperature.
After stirring at room temperature for 18 h, the mixture was
concentrated in vacuo. The residue was poured into water and
extracted with EtOAc. The separated organic phase was successively
washed with water and brine, dried with MgSO4, and concentrated
in vacuo. The residue was purified by column chromatography on
silica gel (30 g, hexane/EtOAc = 17:3) to give 19a (175 mg, 90%) as
23
a colorless solid. Mp 44–45 ꢀC; ½ꢂꢂD ꢁ6:30 (c 1.01, CHCl3). IR ꢁmax
(2S,3R,4E)-2-[(300-Stearoyloxy)triacontanamido]octadec-4-ene-1,3-
diol (2). To a stirred solution of 20a (185 mg, 0.135 mmol) in THF
(5 mL), a solution of tetrabutylammonium fluoride (TBAF, 1.0 M in
THF, 405 mL, 0.405 mmol) was added at room temperature. After
stirring at room temperature for 24 h, the mixture was concentrated
in vacuo. The residue was purified by column chromatography on
silica gel (6 g, CHCl3/MeOH = 25:1) to give a colorless solid. The
obtained solid was washed with Et2O, and then collected by filtration
(KBr) cmꢁ1: 3440 (w, NH), 3320 (br m, OH), 1680 (w, C=C), 1650
(br s, C=O),1590 (w), 1545 (br m), 1250 (m, t-Bu, Si-Me), 1115 (s, C–
O), 1070 (br s, C–O), 970 (w), 840 (br s), 780 (s), 760 (s), 700 (s);
1H-NMR ꢀH (500 MHz, CDCl3): ꢁ0:02 (3H, s), ꢁ0:01 (3H, s), 0.82
(9H, s), 0.88 (3H, t, J ¼ 7:0 Hz), 1.07 (9H, s), 1.21–1.39 (72H, m),
1.50–1.62 (5H, m), 1.95 (2H, quint., J ¼ 7:0 Hz), 1.96–2.04 (2H, m),
3.64 (2H, t, J ¼ 7:0 Hz), 3.70 (1H, dd, J ¼ 4:5, 11 Hz), 3.88 (1H,
dd, J ¼ 5:0, 11 Hz), 4.01 (1H, m), 4.30 (1H, t, J ¼ 6:5 Hz), 5.34
(1H, dd, J ¼ 6:5, 15 Hz), 5.44 (1H, d, J ¼ 9:0 Hz), 5.58 (1H, dt,
J ¼ 15, 7.0 Hz), 7.35–7.44 (6H, m), 7.63 (4H, t, J ¼ 8:0 Hz). HRMS
(ESI+): calcd. for C70H128NO4Si2 [ðM þ HÞþ], 1102.9382; found,
1102.9384.
25
to give 2 (110 mg, 80%) as colorless powder. Mp 94–95 ꢀC; ½ꢂꢂD
ꢁ25:6 (c 0.21, pyridine). IR ꢁmax (KBr) cmꢁ1: 3360 (br m, NH, OH),
1740 (s, C=O), 1620 (br s, C=O), 1540 (br m), 1070 (m, C–O); 1H-
NMR ꢀH (500 MHz, pyridine-d5, 50 ꢀC): 0.880 (3H, t, J ¼ 7:0 Hz),
0.884 (3H, t, J ¼ 7:0 Hz), 1.22–1.44 (100H, m), 1.66 (2H, quint.,
J ¼ 7:0 Hz), 1.71 (2H, quint., J ¼ 7:0 Hz), 1.78–1.87 (2H, m), 2.10
(2H, dt, J ¼ 6:0, 7.0 Hz), 2.40 (2H, t, J ¼ 7:0 Hz), 2.43 (2H, t,
J ¼ 7:0 Hz), 4.20 (2H, t, J ¼ 7:0 Hz), 4.24 (1H, dd, J ¼ 5:0, 11 Hz),
4.40 (1H, dd, J ¼ 5:5, 11 Hz), 4.66 (1H, dddd, J ¼ 5:0, 5.5, 7.0,
8.0 Hz), 4.80 (1H, br s), 5.96 (1H, dt, J ¼ 6:0, 15 Hz), 6.01 (1H, dd,
J ¼ 6:0, 15 Hz), 6.04 (1H, br s), 6.40 (1H, br s), 7.97 (1H, d,
J ¼ 8:0 Hz); 13C-NMR ꢀC (126 MHz, pyridine-d5, 50 ꢀC): 14.3, 23.0,
25.5, 26.3, 26.5, 29.2, 29.5, 29.57, 29.62, 29.63, 29.7, 29.79, 29.81,
29.87, 29.90, 29.93, 29.96, 29.99, 30.03, 30.05, 30.07, 32.2, 32.8, 34.6,
37.0, 57.0, 62.4, 64.5, 73.6, 132.3, 132.4, 173.56, 173.60. HRMS
(2S,3R,4E,6R)-1,3,6-Tris-O-(tert-butyldimethylsilyl)-2-[(300-hydroxy)-
triacontanamido]octadec-4-ene-1,3,6-triol (19b). In the same manner
as that just described, 18b (240 mg, 0.208 mmol) was converted to 19b
24
(211 mg, 91%) as a colorless solid. Mp 33–35 ꢀC; ½ꢂꢂD ꢁ1:24 (c
1.04, CHCl3). IR ꢁmax (KBr) cmꢁ1: 3420 (w, NH), 3280 (br m, OH),
1675 (w, C=C), 1650 (br s, C=O), 1545 (w), 1255 (s, t-Bu, Si-Me),
1080 (br s, C–O), 975 (m), 840 (s), 775 (s); 1H-NMR ꢀH (500 MHz,
CDCl3): 0.01 (3H, s), 0.02 (3H, s), 0.036 (6H, s), 0.043 (3H, s), 0.05
(3H, s), 0.880 (3H, t, J ¼ 7:0 Hz), 0.884 (9H, s), 0.887 (9H, s), 0.891
(9H, s), 1.21–1.38 (70H, m), 1.40–1.52 (2H, m), 1.54–1.63 (4H, m),
2.13 (2H, dt, J ¼ 2:0, 7.5 Hz), 3.54 (1H, dd, J ¼ 5:5, 11 Hz), 3.64 (2H,
t, J ¼ 6:5 Hz), 3.78 (1H, dd, J ¼ 5:5, 11 Hz), 3.98 (1H, m), 4.12 (1H,
dt, J ¼ 5:5, 6.0 Hz), 4.42 (1H, t, J ¼ 5:5 Hz), 5.48 (1H, d, J ¼ 8:0 Hz),
5.60 (1H, ddd, J ¼ 1:0, 5.5, 16 Hz), 5.71 (1H, ddd, J ¼ 1:0, 5.5,
16 Hz). HRMS (ESI+): calcd. for C66H137NO5Si3Na [ðM þ NaÞþ],
1130.9702; found, 1130.9691.
(ESI+): calcd. for
1016.9948.
C
66H130NO5 [ðM þ HÞþ], 1016.9949; found,
(2S,3R,4E,6R)-2-[(300-Stearoyloxy)triacontanamido]octadec-4-ene-
1,3,6-triol (4). In the same manner as that just described, 20b (217 mg,
0.158 mmol) was converted to 4 (85 mg, 52%) as colorless powder. Mp
25
109–111 ꢀC; ½ꢂꢂD þ45:7 (c 0.20, pyridine). IR ꢁmax (KBr) cmꢁ1
:
3480 (w, NH), 3320 (br m, NH, OH), 1740 (s, C=O), 1625 (s, C=O),
1550 (br m), 1170 (br m, C–O), 1060 (m, C–O), 960 (w), 760 (m); 1H-
NMR ꢀH (500 MHz, pyridine-d5, 50 ꢀC): 0.88 (6H, t, J ¼ 7:0 Hz),
1.20–1.43 (98H, m), 1.58 (1H, m), 1.62–1.69 (3H, m), 1.69–1.76 (2H,
m), 1.76–1.85 (2H, m), 2.40 (2H, t, J ¼ 7:0 Hz), 2.41 (2H, t,
J ¼ 7:0 Hz), 4.20 (2H, t, J ¼ 7:0 Hz), 4.24 (1H, dd, J ¼ 5:0, 11 Hz),
4.39 (1H, dd, J ¼ 5:0, 11 Hz), 4.46 (1H, dt, J ¼ 5:5, 6.0 Hz), 4.68 (1H,
m),4.93 (1H, t, J ¼ 5:5 Hz), 6.31 (1H, ddd, J ¼ 1:0, 5.5, 16 Hz), 6.38
(1H, ddd, J ¼ 1:0, 5.5, 16 Hz), 8.01 (1H, d, J ¼ 8:0 Hz); 13C-NMR ꢀC
(126 MHz, pyridine-d5, 50 ꢀC): 14.3, 23.0, 25.5, 26.2, 26.36, 26.40,
29.2, 29.5, 29.59, 29.63, 29.65, 29.83, 29.84, 29.86, 29.88, 29.92,
29.94, 29.97, 29.98, 30.00, 30.04, 30.05, 30.07, 30.08, 30.11, 30.2,
32.2, 34.6, 37.0, 38.7, 57.1, 62.3, 64.5, 71.8, 73.3, 131.1, 136.7, 173.6,
173.7. HRMS (ESI+): calcd. for C66H130NO6 [ðM þ HÞþ], 1032.9898;
found, 1032.9880.
(2S,3R,4E)-3-O-(tert-Butyldimethylsilyl)-1-O-(tert-butyldiphenylsilyl)-
2-[(300-stearoyloxy)triacontanamido]octadec-4-ene-1,3-diol (20a). To
a stirred solution of 19a (71 mg, 0.064 mmol) and pyridine (26 mL,
0.32 mmol) in CHCl3 (5 mL), stearoyl chloride (50 mg, 0.165 mmol)
was added at 0 ꢀC. After stirring at room temperature for 45 min, the
reaction was quenched with water, and the mixture was extracted with
EtOAc. The organic phase was successively washed with water, a
saturated aqueous NaHCO3 solution and brine, dried with MgSO4, and
concentrated in vacuo. The residue was purified by column chroma-
tography on silica gel (10 g, hexane/EtOAc = 10:1) to give 20a
23
(79 mg, 91%) as a colorless solid. Mp 44–45 ꢀC; ½ꢂꢂD ꢁ4:40 (c 1.26,
CHCl3). IR ꢁmax (KBr) cmꢁ1: 3310 (br w, NH), 1740 (s, C=O), 1650
(br m, C=O), 1260 (m, t-Bu, Si-Me), 1175 (s, C–O), 1100 (br s, C–O),
970 (w), 840 (br m), 780 (s), 700 (m); 1H-NMR ꢀH (500 MHz, CDCl3):
ꢁ0:02 (3H, s), ꢁ0:01 (3H, s), 0.83 (9H, s), 0.88 (6H, t, J ¼ 7:0 Hz),
1.07 (9H, s), 1.22–1.36 (100H, m), 1.53 (2H, quint., J ¼ 7:0 Hz), 1.61
(4H, quint., J ¼ 7:0 Hz), 1.95 (2H, q, J ¼ 7:0 Hz), 1.98–2.04 (2H, m),
2.29 (2H, t, J ¼ 7:0 Hz), 3.70 (1H, dd, J ¼ 5:0, 11 Hz), 3.88 (1H, dd,
J ¼ 5:5, 11 Hz), 4.01 (1H, m), 4.05 (2H, t, J ¼ 7:0 Hz), 4.30 (1H, br t,
J ¼ 7:0 Hz), 5.34 (1H, br dd, J ¼ 7:0, 15 Hz), 5.43 (1H, d,
J ¼ 9:0 Hz), 5.58 (1H, br dt, J ¼ 15, 7.0 Hz), 7.35–7.46 (6H, m),
7.64 (4H, t, J ¼ 8:0 Hz). HRMS (ESI+): calcd. for C88H162NO5Si2
[ðM þ HÞþ], 1369.1992; found, 1369.1991.
Acknowledgments
The authors thank Prof. M. Taniguchi (Director,
RIKEN Research Center for Allergy and Immunology)
for his kind support. We are grateful to Dr. S. Hamanaka
(Hamanaka Dermatological Clinic) for her helpful
suggestion. Our thanks are due to Drs. T. Nakamura
and Y. Hongo (RIKEN) for the HRMS analysis. We also
thank Dr. Y. Masuda (T. Hasegawa Co., Ltd.) for
discussions. Dr. H. Matsuda of Takasago International
Corporation kindly provided 15-pentadecanolide.
(2S,3R,4E,6R)-1,3,6-Tris-O-(tert-butyldimethylsilyl)-2-[(300-stearoy-
loxy)triacontanamido]octadec-4-ene-1,3,6-triol (20b). In the same
manner as that just described, 19b (192 mg, 0.173 mmol) was
converted to 20b (233 mg, 98%) as a colorless solid. Mp 43–44 ꢀC;

1720
T. TASHIRO and K. MORI
13) Matsuda H and Mori K, Biosci. Biotechnol. Biochem., 56, 2076–
2078 (1992).
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